The in vitro isolation and analysis of pancreatic stem/progenitor cells are necessary for understanding their properties and function; however, the preparation of high-quality single-cell suspensions from adult pancre...The in vitro isolation and analysis of pancreatic stem/progenitor cells are necessary for understanding their properties and function; however, the preparation of high-quality single-cell suspensions from adult pancreas is pre- requisite. In this study, we applied a cold trypsin-ethylenediaminetetraacetic acid （EDTA） digestion method to disas- sociate adult mouse pancreata into single cells. The yield of single cells and the viability of the harvested cells were much higher than those obtained via the two commonly used warm digestion methods. Flow cytometric analysis showed that the ratio of ductal or BCRPl-positive cells in cell suspensions prepared through cold digestion was con- sistent with that found in vivo. Cell culture tests showed that pancreatic epithelial cells prepared by cold digestion maintained proliferative capacity comparable to those derived from warm collagenase digestion. These results indicate that cold trypsin-EDTA digestion can effectively disassociate an adult mouse pancreas into viable single cells with minimal cell loss, and can be used for the isolation and analysis of pancreatic stem/progenitor cells.展开更多
Although the role of copper (Cu) in promoting KRas- or BRaf-mutation driven cancers via activating MEK1/2 kinases is known, the mechanism by which the copper transporter SLC31A1 (CTR1) is upregulated in pancreatic can...Although the role of copper (Cu) in promoting KRas- or BRaf-mutation driven cancers via activating MEK1/2 kinases is known, the mechanism by which the copper transporter SLC31A1 (CTR1) is upregulated in pancreatic cancer (PDAC, KRas mutation) is not defined. In this study, we provide evidence that MEK signal maintains a high level of SLC31A1 through silencing the expression of miR-124-3P (miR-124) via a novel MEK-DNMT1-miR-124 feedback loop in PDAC cells. Further, we reveal that miR-124 directly targets suppression of SLC31A1, and miR-124 introduction together with tetrathiomolybdate (TM) treatment hampered pancreatic cancer growth in vitro and in vivo. Our results demonstrate that a SLC31A1-MEK-DNMT1-miR-124 feedback loop is an important pathway to maintain copper absorption and promote pancreatic cancer progression, and we hope to provide a Cu-chelation as an adjuvant treatment strategy, to block the progression in Kras mutant PDAC patients.展开更多
基金supported by the National Natural Science Foundation of China (No. 31272520)the Special Fund for Scientific and Technological Innovation Talents in Harbin, China (No. 2012RFXXS048)
文摘The in vitro isolation and analysis of pancreatic stem/progenitor cells are necessary for understanding their properties and function; however, the preparation of high-quality single-cell suspensions from adult pancreas is pre- requisite. In this study, we applied a cold trypsin-ethylenediaminetetraacetic acid （EDTA） digestion method to disas- sociate adult mouse pancreata into single cells. The yield of single cells and the viability of the harvested cells were much higher than those obtained via the two commonly used warm digestion methods. Flow cytometric analysis showed that the ratio of ductal or BCRPl-positive cells in cell suspensions prepared through cold digestion was con- sistent with that found in vivo. Cell culture tests showed that pancreatic epithelial cells prepared by cold digestion maintained proliferative capacity comparable to those derived from warm collagenase digestion. These results indicate that cold trypsin-EDTA digestion can effectively disassociate an adult mouse pancreas into viable single cells with minimal cell loss, and can be used for the isolation and analysis of pancreatic stem/progenitor cells.
基金supported by The National Natural Science Foundation of China(No.32072801)Fundamental Research Funds for the Central Universities of China(No.2572020DY12).
文摘Although the role of copper (Cu) in promoting KRas- or BRaf-mutation driven cancers via activating MEK1/2 kinases is known, the mechanism by which the copper transporter SLC31A1 (CTR1) is upregulated in pancreatic cancer (PDAC, KRas mutation) is not defined. In this study, we provide evidence that MEK signal maintains a high level of SLC31A1 through silencing the expression of miR-124-3P (miR-124) via a novel MEK-DNMT1-miR-124 feedback loop in PDAC cells. Further, we reveal that miR-124 directly targets suppression of SLC31A1, and miR-124 introduction together with tetrathiomolybdate (TM) treatment hampered pancreatic cancer growth in vitro and in vivo. Our results demonstrate that a SLC31A1-MEK-DNMT1-miR-124 feedback loop is an important pathway to maintain copper absorption and promote pancreatic cancer progression, and we hope to provide a Cu-chelation as an adjuvant treatment strategy, to block the progression in Kras mutant PDAC patients.
